WO1999032658A1

WO1999032658A1 - Overlapping nucleotide sequences, and their use for detecting xenohormones and in pharmaceutical compositions

Info

Publication number: WO1999032658A1
Application number: PCT/FR1998/002793
Authority: WO
Inventors: Robert Barouki; Michèle GARLATTI-VINCENT; Charbel Massaad
Original assignee: Inserm
Priority date: 1997-12-18
Filing date: 1998-12-18
Publication date: 1999-07-01
Also published as: FR2772786A1

Abstract

The invention concerns the use of nucleotide sequences comprising one or several units responsive to glucocorticoids, mineralocorticoids, androgens and progestogens (also referred to as GRU), and/or one or several estrogen-responsive units (also referred to as ERU), said responsive units being characterised in that their two response elements overlap each other such that the centre of one of these two elements is offset by a distance of five base pairs relative to the centre of the other of these two elements, for implementing a method for detecting in vitro xenohormones, or in preparing medicines for treating pathologies related to the activity of glucocorticoid, mineralocorticoid, androgen and progestogen receptors, and/or estrogen receptors.

Description

OVERLAP NUCLEOTIDE SEQUENCES, AND THEIR USE IN THE CONTEXT OF DETECTION OF XENOHORMONES AND IN PHARMACEUTICAL COMPOSITIONS

The subject of the present invention is the use of nucleotide sequences derived from the glucocorticoid and / or estrogen response elements, for the implementation of methods of in vitro detection of xenohormones.

A subject of the invention is also pharmaceutical compositions containing such nucleotide sequences.

The toxicity of many compounds found in the environment as well as of a certain number of drugs is due to their interaction with hormone receptors. Such compounds are also designated xenohormones.

The illegitimate activation of these hormone receptors can have various consequences on cell metabolism, differentiation and / or proliferation.

A typical example is that of pesticides and certain air pollutants which, by activating the estradiol receptor, will trigger an estrogenic response.

These xenoestrogens have been suspected to be responsible for the decline in male fertility and the increase in the frequency of certain cancers in recent years such as testicular cancer and breast cancer (Auger et al.; Jensen et al., 1995).

Other compounds seem to have glucocorticoid-like effects, and could thus lead to disturbances of the immune system (Tanaka et al., 1996).

Xenohormones having very diverse structures, it is difficult to propose a direct structural assay. A functional assay can be considered in animals using the physiological properties of activated receptors, but this approach would be too cumbersome and too expensive if it were to be used routinely.

We can therefore focus on cell systems in culture, in particular using cell transfections (the different approaches are discussed in Sonnenschein et al., 1995).

The principle consists in cloning a promoter regulated by a given nuclear receptor upstream of a reporter gene coding for an enzyme whose assay is simple and sensitive. If the cell has the receptor in question, the addition of xenohormone will activate the promoter which would be revealed by the reporter gene.

There are several variants of this approach using either mammalian cells or yeasts.

The promoters used are natural promoters, for example that of the vitellogenin gene for estrogens and that of the MMTV virus (Mouse Mammary & Tumor Virus) for glucocorticoids.

Despite their interest, these promoters have a certain number of drawbacks: i) they are not necessarily the most sensitive to the receptor studied, ii) they can be regulated by other effectors; indeed, genes are often subject to multiple regulations and it is frequent that these regulations are colocalized in the promoter in a regulatory unit (or regulatory domain) (Lucas and Granner, 1992).

The use of these promoters can therefore pose a problem of specificity of the effect observed.

Nuclear receptors are the intermediaries at the cellular level of the action of several hormones and effectors such as steroid hormones, triodothyronine, retinoic acid, and 1-25 (OH) 2 vitamin D3 (Evans, 1988).

These hormonal receptors take on such a configuration, when they are linked to hormones, that they then recognize DNA sequences designated as response elements to said receptors, and that they thus activate the various promoters comprising said response elements.

There are two main families of nuclear receptors activated by a ligand (Beato et al., 1995; Mangelsdorf et al., 1995):

- the family of glucocorticoid, mineralocorticoid, androgen and progestin receptors (also called glucocorticoid receptors),

- the family of estrogen and triodothyronine receptors.

Most receptors of the latter family bind DNA in the form of heterodimers with the cis-retinoic acid receptor, RXR. Their response elements generally consist of direct repetitions of the AGGTCA half-site (Mangelsdorf and Evans, 1995). The element of response to estrogens is, interestingly, different in that it is usually palindromic and formed of two inverted half-sites separated by three base pairs (Klein-Hitpaβ et al., 1988a). This structure is also that of the response elements of the glucocorticoid receptor family (Martinez et al., 1987), but in this case, the sequence of the half-sites is different.

Nuclear receptors have a common domain structure with two highly conserved regions: the central DNA binding domain and the C-terminal hormone binding domain (Kumar et al., 1987; Fawell et al., 1990 ; Kumar et al., 1986; Tora et al., 1989; Tasset et al., 1990; Webster et al., 1988; Lees et al., 1989). After hormonal stimulation, the estrogen receptor (ER), binds an element of response to estrogens, ERE, in the form of a homodimer thus modulating the genetic expression (Brandt et al., 1997; Kumar et al., 1988; Beato, 1989; Dean et al., 1996).

The efficiency and specificity of the regulation of a gene by a hormone depends on the sequence, the orientation and the spacing of the half-sites but also on the number and the localization of the elements of response (Kraus et al ., 1994; Anolik et al., 1995; Discroll et al., 1996). Indeed, a characteristic common to a large number of promoters regulated by hormones is the presence of several adjacent response elements. In many cases, these elements have a synergistic effect on the transcription of the gene (Klein-Hitpaβ et al., 1988b; Martinez et al., 1989). It is also known that in the case of certain nuclear receptors and transcription factors, a cooperative bond is observed on adjacent elements (Martinez et al., 1989; Tsai et al., 1989). The distance between these elements is critical. It becomes optimal when it corresponds to a multiple of helix turns which then allows the binding of receptors on the same side of the DNA double helix. However, this distance can also be very long. In this case, the loop formed by DNA would allow interaction between receptors and stabilization of the complex (Ptashne, 1986). The flexibility of the protein would be critical for such interactions to occur.

A glucocorticoid response unit, also referred to hereinafter as GRE A, containing two elements of response to glucocorticoids arranged in an overlapping manner has been demonstrated in the context of work on the regulation of the promoter of the cytosolic aspartate aminotransferase gene ( AspATc) (Garlatti et al., 1994). This sequence is recognized by a receptor tetramer, which forms in a highly cooperative manner. Work to modify this GRE A sequence has been carried out in order to increase its affinity for the tetramer, and has resulted in the sequence designated below GRE A up (Garlatti et al., 1994).

However, no correlation between, on the one hand, the highlighting of these GRE A and GRE A up response units mentioned above, and, on the other hand, the The possibility of using such overlapping response elements in the context of implementing a method for detecting xenohormones has not been made, nor even suggested in the article by Garlatti et al mentioned above.

Indeed, the aim of this article was that of the study, on a fundamental level, of a promoter, namely the promoter of the AspATc gene, and of the glucocorticoid response element activating this promoter, when said element of response is recognized by glucocorticoid receptors.

The glucocorticoid used in the activation experiments of the promoter of the AspATc gene described in the article Garlatti et al. mentioned above, is dexamethasone, namely a synthetic analogue of cortisone. Unlike xenohormones likely to be found in the environment, dexamethasone is a very powerful agonist of glucocorticoid receptors, and as such represents the most effective synthetic analogue of cortisone.

Consequently, no correlation has been made or suggested in this article, between, on the one hand, the GRE A and GRE A up response units mentioned above, and, on the other hand, the use of these units for the detection of xenohormones in samples, such as water or plant or soil extracts, as there was nothing to suggest that the amounts of xenohormones present in such samples were likely to be sufficient to activate promoters by above response units.

The present invention follows from the discovery by the Inventors:

- on the one hand, the fact that the elements for responding to estrogens can also be arranged artificially so as to overlap within the framework of an estrogen response unit, as had been highlighted above in the case of a natural glucocorticoid response element, namely the GRE A element, and this despite the differences, both in their amino acid sequences and in their three-dimensional structure, existing between the estrogen response elements , and those with glucocorticoids,

- and, secondly, the fact that the glucocorticoid and estrogen response units, comprising overlapping response elements, make it possible to detect xenohormones at doses up to approximately 10 to 100 times weaker than the allow the corresponding non-overlapping response elements.

One of the aims of the present invention is to provide new methods for detecting xenohormones present in the environment or in the body. human or animal, or screening methods for compounds capable of being xenohormones, these methods being simple to use and more specific for said xenohormones than the existing methods of the prior art.

The present invention also aims to provide new pharmaceutical compositions which can be used in the treatment of pathologies wholly or partly associated with the presence of such xenohormones in the environment, and therefore with the activity of glucocorticoid and mineralocorticoid receptors, androgens and progestins, and / or estrogen receptors.

Another object of the present invention is to provide new response units capable of being used in a detection method as described above, or in the abovementioned pharmaceutical compositions.

The subject of the present invention is the use of nucleotide sequences comprising one or more glucocorticoid, mineralocorticoid, androgen and progestin response units (also called glucocorticoid response units, or GRU), and / or one or more response units to estrogens (also called ERU), said units being such that they include:

- for the GRU unit: two nucleotide sequences capable of activating the promoters by the glucocorticoids, when they are recognized by the receptors associated with the latter, these two sequences, also known as glucocorticoid response elements (or GRE elements), being sequences derived by modification of one or more nucleotides of the GRE response element corresponding to the DNA sequence of the following formula:

⁵ 'GGTACA X! X _{2 3} TGTTCT ³ '

₃ , CCATGT X ₄ X ₅ X ₆ ACAAGA ₅ , in which Xi, X2, and X3, independently of each other, represent any nucleotide, while X4, X5, and Xg, represent a nucleotide capable of pairing with Xi, X2, and X3, respectively,

- for the ERU unit: two nucleotide sequences capable of activating the promoters by estrogens, when they are recognized by the receptors associated with the latter, these two sequences, also known as elements of response to estrogens (or ERE elements), being sequences derived by modification of one or more nucleotides of the GRE response element corresponding to the DNA sequence of the following formula:

⁵ 'AGGTCA X _! X ₂ X ₃ TGACCT ³ ' ₃ , TCCACT XX ₅ X ₆ ACTGGA ₅ , in which Xi, X, and X3, independently of each other, represent any nucleotide, while X4, X5, and Xg, represent a nucleotide capable of pairing with Xi, X, and X3, respectively, said units of response GRU and ERU being characterized in that their two respective response elements, GRE and ERE, overlap in such a way that the center of one of the elements GRE or ERE, is respectively offset by a distance of 5 base pairs relative to the center of the other GRE or ERE element, for the implementation of a method of in vitro detection of xenohormones, or for the preparation of medicaments intended for the treatment of pathologies associated with the activity of glucocorticoid receptors and / or estrogen.

The expression "glucocorticoid response element" means any nucleotide sequence capable of activating the promoters by the glucocorticoids, when they are recognized by the receptors associated with the latter.

By the expression "element of response to estrogens" is meant any nucleotide sequence capable of activating the promoters by estrogens, when they are recognized by the receptors associated with the latter.

Advantageously, the modifications made to the GRE or ERE response elements, the formulas of which are given above, are essentially such that they allow said response units to activate the promoters comprising them, when these units are recognized by the associated receptors to glucocorticoids, mineralocorticoids, androgens and progestins, or by estrogen receptors.

The invention relates more particularly to the abovementioned use of nucleotide sequences comprising, as GRU unit:

the nucleotide sequence of formula (I) below:

XalGT ACX _bl GX _ci AX _dl GX _el CCX _fl GTT CX _gX ₃ , X _a2 CA τGX _b2 CX _c2 TX _d 2C e2 GGX _f2 CAA GX _{g2 g ι}

in which :

. the GRE response elements are indicated, on one of the DNA strands, in bold and italic type,

. X _a ι to Xgi, independently of each other, represent any nucleotide or derivative, while X _a 2 to Xg2 _* represent a nucleotide or derivative capable of pairing with X _a ι to X _ε ι respectively, - or a succession of at least two nucleotide sequences of formula (I) above, each of the nucleotide sequences of formula (I) being spaced by a distance between lOn and l in base pairs, center to center, n being a whole number greater than or equal to 2.

Advantageously, the nucleotide sequences comprising, as GRU unit, the nucleotide sequence of formula (I), or a succession of at least two nucleotide sequences of formula (I), mentioned above, are characterized in that:

- X _a ι, X _D ι, Xfi, and Xgi, independently of each other, represent any nucleotide or derivative, while X _a 2, X _D 2 _' Xf2 _> ^and g2 _> represent a nucleotide or derivative capable of s '' pair with X ^, X51, Xfi, and Xgi, respectively,

- X _c ι represents A or T, while X _c 2 represents T or A respectively,

- Xdi represents A or C, while X ^ β represents T or G respectively,

- X _e ι represents A or T, while X _e 2 represents T or A respectively.

A more particular subject of the invention is the abovementioned use of nucleotide sequences comprising, as GRU unit:

- the following nucleotide sequence designated "GRE A":

⁵ 'GGT ACA GAA AGA CCT GTT CT ³ ' 3. CCA TGT CTT TCT GGA CAA GA 5.

in which the GRE response elements are indicated, on one of the strands of DNA, in bold type, italics, and underlined,

- or a succession of at least two GRE A nucleotide sequences, spaced by a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (GRE A) 2:

'' GGT ACA GAA AGA CCT GTT CTX _a GGT ACA GAA AGA CCT GTT CT ³ 1 CCA TGT CTT TCT GGA CAA GAX _b CCA TGT CTT TCT GGA CAA GA ₅

in which X _a represents any nucleotide or derivative, and X represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X5 represents G, . the following sequence (GRE A) 4: ⁵ 'GGT ACA GAA AGA CCT GTT CTX _a GGT ACA GAA AGA CCT GTT CT ³ ' ₃ . CCA TGT CTT TCT GGA CAA GAX _h CCA TGT CTT TCT GGA CAA GA ₅ . ⁵ 'X _C GGT ACA GAA AGA CCT GTT CTX _e GGT ACA GAA AGA CCT GTT CT ³ ' ₃ ι X _d CCA TGT CTT TCT GGA CAA GAX _f CCA TGT CTT TCT GGA CAA GA ₅ ,

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X _D , X, and Xf, represent a nucleotide or derivative capable of pairing with X _a , X _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

A more particular subject of the invention is also the abovementioned use of nucleotide sequences comprising, as GRU unit:

- the following nucleotide sequence designated "GRE A up":

5 'GGT ACA GTA CGT CCT GTT CT 3'

3 'CCA TGT CAT GCA GGA CAA GA 5'

- or a succession of at least two GRE A up nucleotide sequences, spaced by a distance between lOn and lin base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (GRE A up) 2:

⁵ 'GGT ACA GTA CGT CCT GTT CTX _a GGT ACA GTA CGT CCT GTT CT ³ ' ₃ ι CCA TGT CAT GCA GGA CAA GAX _b CCA TGT CAT GCA GGA CAA GA 5.

in which X _a represents any nucleotide or derivative, and X5 represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X5 represents G,

. the following sequence (GRE A up) 4:

⁵ 'GGT ACA GTA CGT CCT GTT CTX _a GGT ACA GTA CGT CCT GTT CT ³ '

₃ . CCA TGT CAT GCA GGA CAA GAX _h CCA TGT CAT GCA GGA CAA GA 51

⁵ 'X _C GGT ACA GTA CGT CCT GTT CTX _e GGT ACA GTA CGT CCT GTT CT ³

₃ 1 X _d CCA TGT CAT GCA GGA CAA GAX _£ CCA TGT CAT GCA GGA CAA GA 5 in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X, X <j, and Xf, represent a nucleotide or derivative capable of pairing with X _a , X _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

The invention also relates to the abovementioned use of nucleotide sequences comprising, as ERU unit:

the nucleotide sequence of formula (II) below:

X _al GG TCX _hl GGT CGA CCX _cl GAC CX _d ι X _a2 CC AGX _b2 CCA GCT GGX _c2 CTG GX _d2

in which :

. the ERE response elements are indicated, on one of the DNA strands, in bold and italic type,

• Xal to Xdi, independently of each other, represent any nucleotide or derivative, while X _a 2 to X <χ, represent a nucleotide or derivative capable of pairing with X _a ι to XJI respectively,

- or a succession of at least two nucleotide sequences of formula (II) above, spaced by a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2.

A more particular subject of the invention is the abovementioned use of nucleotide sequences comprising, as ERU unit:

- the following nucleotide sequence designated "over ERE":

⁵ 'AGG TCA GGT CGA CCT GAC CT ³ ' 3. TCC AGT CCA GCT GGA CTG GA 51

in which the ERE response elements are indicated, on one of the strands of DNA, in bold, italic and underlined characters,

- or a succession of at least two nucleotide sequences over ERE, spaced by a distance between lOn and lin base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (over ERE) 2:

'AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ ' 1 TCC AGT CCA GCT GGA CTG GAX _b TCC AGT CCA GCT GGA CTG GA 5. in which X _a represents any nucleotide or derivative, and X represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X1 represents G,

. the following sequence (over ERE) 4:

'AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ ' 1 TCC AGT CCA GCT GGA CTG GAX _h TCC AGT CCA GCT GGA CTG GA 5. 'X _C AGG_TÇA GGT CGA CCT GAC CTX _e AGG TCA GGT CGA CCT GAC CT ³ '1 X _d TCC AGT CCA GCT GGA CTG GAX _f TCC AGT CCA GCT GGA CTG GA 5.

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X, X <- \, and Xf, represent a nucleotide or derivative capable of pairing with X _a , X _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

The subject of the invention is also any nucleotide sequence characterized in that it comprises a succession of at least two GRE A nucleotide sequences, and / or a succession of at least two GRE A up nucleotide sequences, as defined above. above.

The invention also relates to any nucleotide sequence characterized in that it comprises, as ERU unit:

the nucleotide sequence of formula (II) below:

X _al GG TCX _bl GGT CGA CCX _cl GAC CX _dl ₃ , Xa2CC AGXi CCA GCT GGX _c2 CTG GX _{d2 gι}

in which :

. X _a ι to Xdi, independently of each other, represent any nucleotide or derivative, while X _a 2 to Xd2 _> represent a nucleotide or derivative capable of pairing with X _a j to X ^ i respectively,

The invention more particularly relates to any nucleotide sequence as described above, and characterized in that it comprises, as ERU unit: - the following nucleotide over ERE sequence:

⁵ 'AGG TCA GGT CGA CCT GAC CT ³ ' 3 ι TCC AGT CCA GCT GGA CTG GA 5.

- or a succession of at least two nucleotide sequences over ERE, spaced by a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (over ERE) 2:

'AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ ' 1 TCC AGT CCA GCT GGA CTG GAX _h TCC AGT CCA GCT GGA CTG GA 51

. the following sequence (over ERE) 4:

'AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ ' 1 TCC AGT CCA GCT GGA CTG GAX _h TCC AGT CCA GCT GGA CTG GA 51 'X _ç AGG TCA GGT CGA CCT GAC CTX _e AGG TCA GGT CGA CCT GAC CT ³ 1 X _d TCC AGT CCA GCT GGA CTG GAX _f TCC AGT CCA GCT GGA CTG GA ₅

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X5, X ^, and Xf, represent a nucleotide or derivative capable of pairing with X _a , X _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

The subject of the invention is also any nucleotide sequence comprising one or more ERU units, as defined above, and also comprising one or more GRU units defined above.

The invention also relates to the nucleotide sequences defined above, in which the ERU unit (s), and, where appropriate the GRU unit (s), are located upstream of a basal promoter controlling the transcription of a reporter gene. Advantageously, the basai promoter is chosen from the following:

- the ΔMTV promoter corresponding to the promoter of the deleted MMTV virus, described in Umesono et al. , 1988, Nature, 336, 262-265,

the TK promoter corresponding to the promoter of the viral thymidine kinase,

- the promoter reduced to the TATA box, corresponding to a fragment of the promoter of the viral thymidine kinase.

Advantageously also, the reporter gene is chosen from the following genes:

- the gene coding for luciferase,

- the gene coding for β-galactosidase,

- the gene coding for chloramphenicol acetyl transferase,

- the gene coding for the cytochrome P450 1A1 metabolizing ethoxycoumarin into fluorescent hydroxycoumarin, capable of being detected directly in the culture medium,

- the gene coding for cytochrome P450 2A6 metabolizing coumarin into fluorescent hydroxycoumarin, capable of being detected directly in the culture medium.

The subject of the invention is also any vector, in particular any plasmid, characterized in that it comprises at least one nucleotide sequence as described above according to the invention.

The subject of the invention is more particularly any vector, in particular any plasmid, characterized in that it comprises at least one nucleotide sequence comprising an ERU unit, as described above according to the invention.

A vector particularly preferred in the context of the present invention is that containing, as ERU unit, the over ERE sequence described above, or a succession of at least two over ERE sequences.

The invention also relates to any human or animal cell transformed by a vector as defined above.

Preferably, the above cells are chosen from the following cells (referenced in the ATCC catalog: American Type Culture Collection):

- Hep G2, Hep 3B, TONG / HCC, human hepatomatous cells,

- CV1 monkey kidney fibroblasts,

- human hepatocytes,

- MCF7 cells of breast carcinoma,

- LNCaP cells of prostatic carcinoma. The invention also relates to any method of detection or in vitro screening of xenohormones capable of interacting with glucocorticoid receptors, and / or estrogen receptors in the human or animal organism, characterized in that it comprises:

the cultivation of human or animal cells as described above, in a culture medium in the presence of a biological sample capable of containing said xenohormones,

- detecting the presence of the polypeptide encoded by said reporter gene in said cells or the culture medium thereof, in particular by colorimetry in the case where the polypeptide encoded by the reporter gene reacts with its substrate in the context of a reaction colored, or by fluorimetry in the case where the polypeptide coded by the reporter gene is fluorescent, or capable of being recognized by a fluorescent molecule, or by chemiluminescence, or even by a radioactive method.

Advantageously, the biological sample likely to contain xenohormones is water, plant extracts, extracts from soil samples, in the case of the implementation of a method for detecting these xenohormones in the environment, or a blood sample in the case of the implementation of a method of in vitro detection of these xenohormones in the human or animal body.

A preferred in vitro detection method according to the invention, of xenohormones capable of interacting with glucocorticoid receptors, is that comprising:

- culturing human or animal cells as described above, transformed with a vector containing, as GRU unit, the GRE A up sequence described above, or a succession of at least two GRE sequences A up, in a culture medium in the presence of a biological sample capable of containing said xenohormones,

- Detecting the presence of the polypeptide encoded by said reporter gene in said cells or the culture medium of the latter, in particular in the manner indicated above.

A preferred in vitro detection method according to the invention, of xenohormones capable of interacting with estrogen receptors, is that comprising:

the cultivation of human or animal cells as described above, transformed with a vector containing, as ERU unit, the over ERE sequence described above, or a succession of at least two over sequences TIME. in a culture medium in the presence of a biological sample capable of containing said xenohormones,

The invention also relates to any pharmaceutical composition characterized in that it comprises, in association with a physiologically acceptable vehicle, at least one GRU and / or ERU unit as defined above.

Preferred pharmaceutical compositions in the context of the present invention are those comprising, as GRU unit, the GRE A up sequence described above, or a succession of at least two GRE A up sequences.

Other pharmaceutical compositions preferred in the context of the present invention are those comprising, as ERU unit, the over ERE sequence described above, or a succession of at least two over ERE sequences.

Advantageously, the abovementioned pharmaceutical compositions of the invention are characterized in that the GRU unit (s), and / or the ERU unit (s) are preceded and followed by a succession of at least about 5 nucleotides or derivatives.

Preferably, the abovementioned pharmaceutical compositions of the invention are presented in a form which can be administered by the parenteral route, in particular by the intravenous route, or by the oral or local route. , in particular at a rate of approximately 0.1 mg / kg to approximately 10 mg / kg per day of treatment.

The subject of the invention is also any vector for gene therapy, characterized in that it comprises at least one GRU and / or ERU unit, as defined above.

Vectors for gene therapy which are particularly preferred in the context of the present invention are those comprising, as GRU unit, the GRE A up sequence described above, or a succession of at least two GRE A up sequences, or ERU unit title, the over ERE sequence described above, or a succession of at least two over ERE sequences.

The invention also relates to any pharmaceutical composition comprising a vector for gene therapy as defined above, in association with a physiologically acceptable vehicle.

The invention also relates to the use of pharmaceutical compositions comprising at least one GRU unit, as described above, in the context of the treatment of pathologies associated with the function of a receptor to steroids, including prostate cancer or Cushing's syndrome.

The invention also relates to the use of pharmaceutical compositions comprising at least one ERU unit, as described above, in the context of the treatment of pathologies associated with the activity of the function of an estrogen receptor, in particular the breast, cervical and ovarian cancers.

A subject of the invention is also the kits or kits for the implementation of a method of in vitro detection of xenohormones, as described above, comprising:

cells, as defined above, transformed with a vector containing one or more GRU and / or ERU units, said cells being advantageously cultured at 37 ° C. and stored at -80 ° C.,

- where appropriate, a culture medium for said cells,

- where appropriate, the reagents necessary for the detection of the polypeptide encoded by the reporter gene.

The invention will be further illustrated with the aid of the following detailed description of the construction of the over ERE sequence, and of its use in the context of the detection of xenohormones.

I) MATERIALS AND METHODS

1) Cell culture

HeρG2 cells (Knowles et al., 1980), MCF-7 cells (breast tumor cells) and CMT cells are maintained in DMEM (Dulbecco's minimal essential medium) medium without phenol red, and containing 10% serum fetal calf (Gibco), penicillin (100 U / ml), streptomycin (100 μl / ml) (Diamond) and fungizone (0.5 μg / ml) (Squibb).

2) Plasmids

The vectors for expression of the human estradiol receptor (ER) as well as the truncated fragments of the receptor (HE13, HE15, HE19, HE21, HE38, HE70, and HE72) have been described by Green et al., 1987, and T. Ylicomi et al. , 1992. The plasmid ΔMTV-CAT derives from the plasmid MMTV-CAT, however the regulatory sequences sensitive to glucocorticoids have been deleted (Umesono et al. 1988). A Hind III site has been created at this position. It will serve as a cloning site for oligonucleotides. The oligonucleotides were also subcloned into the HindIII site of the Tk-CAT plasmid. The oligomers {ERE, (ERE) 2, over ERE, and (over ERE) 2} have 5 'ends compatible with the HindIII site. However, the restriction site is lost after the subcloning. The sequence (ERE) 2 was obtained by the ligation of two ERE oligonucleotides. Sequence of ERE: strand A: ⁵ 'AGCTGCTC AGCT AGGTCA CTG CGACCT CTACT ³ ', strand B: ^{5 '} AGCTAGTAG AGGGCT GAG TGACCT AGCTGAGC ³ ' - Sequence of over ERE: strand A: ⁵ 'AGCTGCTC AGCT AGGTCAGGTCGACCTGACCT CTACT ³ ' : 5 ' _AGCTAG TAG AGGTCAGGTCGACCT GACCT AGCTGAGC ³ '. (Over ERE) sequence 2: strand A: ⁵ 'AGCTGCTC AGCT AGGTCAGGTCGACCTGACCT C AGGTCAGGTCGACCTGACCT CTACT ³ ', strand B: 5 ' _AG CTAGTAG AGGTCAGGTCGACCTGACCT G AGGTCAGGTCGACCTGACCT

AGCTGAGC ³ '. (ERE sites are underlined). The GRE sequence used is derived from the promoter of the cytosolic aspartate aminotransferase gene (Garlatti et al., 1994). This sequence has a transcriptional efficiency comparable to the consensus GRE. The luciferase-expressing plasmid (RSV-Luc) was purchased from Promega.

3) Transient transfection

Transfection experiments have been described by Massaad et al., 1997. One day before transfection, the HepG2 cells are seeded at 10 "cells in a culture dish 10 cm in diameter. The culture medium is replaced with medium. containing no steroids 2 to 3 hours before transfection To remove the steroids, the serum is heated twice for 20 minutes at 65 ° C on activated charcoal trapping the steroids.

In a sterile tube, 5 μg of the plasmid to be studied (ERE-CAT, over ERE-CAT ...), different quantities of the ER expression plasmid (estrogen receptor) and 1 μg of the plasmid coding for luciferase are mixed in a final volume of 500 μl containing 62.5 μl of CaCl2 (2M). This mixture is added dropwise and with bubbling to 500 μl of HBS 2X (Nacl 280 mM, Hepes 50 mM, Na2HPθ4 1.5 mM pH 7.1). This pH is essential for the formation of a stable DNA-calcium phosphate precipitate. Thirty minutes are necessary for the course of the complexing reaction. This mixture is then added dropwise to the cells which are incubated for 3 h 30 min. This incubation promotes the precipitation of DNA and its adhesion to cells. Thereafter, treatment with PBS IX containing 20% by volume of glycerol allows the internalization of the DNA-calcium grains. Two minutes later, 5 ml of PBS IX, pH 7.5, are added in order to dilute the glycerol. A second washing with PBS is necessary to remove all traces of glycerol. Ten ml of complete medium are added to the cells and the dishes are incubated for 24 hours so that the transfected plasmids are expressed.

A similar protocol is used for MCF-7 cells. The cells are seeded at 10 ° ^"cells per 6 cm in diameter and transfected according to the technique calcium phosphate (Jiang et al., 1992).

Sixteen hours later, the medium is replaced by a 5% FCS medium without steroids; then the boxes are treated with the hormones to be tested: estradiol (10 ^{~ 7} M final concentration) antihormone (ICI 164386) or xenohormones. A 24 hour incubation allows hormonal induction.

4) Assay of CAT activity:

Chloramphenicol acetyltransferase ensures the transfer of an acetyl group to the hydroxyl in position 3 of chloramphenicol (antibiotic). The quantification of the acetylated product allows direct measurement of the regulatory activity of a promoter.

The classical technique consists in extracting the product and the substrate, in separating them by chromatography (TLC) and autoradiography. The method we have used, faster, is a transfer of radioactivity of acetyl CoA ⁽³ H or 14 _to ι _e chloramphenicol easily detectable thanks

(g ₎ to a scintillation counter. The scintillating liquid used is econofluor which is hydrophobic. However acetylated chloramphenicol diffuses in the econofluor while acetyl CoA remains in the aqueous phase. Thus, only the product of the reaction is in an environment allowing its detection by the scintillation counter.

Experimental protocol:

After the aspiration of the culture medium, two washes with PBS IX without calcium or magnesium are carried out. Five hundred μl of lysis buffer IX (Promega's Reporter Lysis Buffer) are added to the culture dishes after two rinses with PBS IX without Ca ^ + or Mg2 +. Fifteen minutes later, the cells are detached by scraping and then centrifuged at 8000 g. The sample is separated into 3 fractions: 100 μl will be used for the CAT assay, 20 μl will be used for the luciferase assay and 80 μl will be used for the assay of the proteins.

One hundred μl of supernatant are heated at 65 ° C. for 5 minutes in order to denature the endogenous acetylases, then centrifuged for 20 minutes at 15,000 rpm. Twenty μl of Premix (5 mM EDTA, 250 mM Tris-HCl pH 7.5, cold acetyl CoA 30 μM, ³ H acetyl CoA 0.5 μCi, chloramphenicol ImM) and 20 μl of chloramphenicol (5 mM) are added to 60 μl samples. The samples are then incubated for 30 minutes at 37 ° C. Eighty μl of this mixture are added to 4 ml of Econofluor '. A vigorous mixing allows the diffusion of chloramphenicol towards the hydrophobic phase; then the samples are incubated for 30 minutes at room temperature; this allows the separation of the two phases and the diffusion of radioactivity towards the Econofluor ^® where the counting is carried out. Radioactivity is measured using the Beckman scintillation counter (model: LS 5000 CE). It should be noted that the diffusion of radioactivity is a linear function of time (Neumann et al., 1987).

5) Determination of luciferase:

The dosage of luciferase is extremely sensitive, fast and simple. Luciferase is a monomeric protein with a molecular weight of 62 KDa. It catalyzes the oxidation of luciferin (peroxide) accompanied by the production of photons.

The conventional luciferase test generates a brief flash of light which appears upon contact of the enzyme with the substrate. But the system we used (Promega's luciferase assay system) has a slower enzymatic "turnover" allowing to visualize a constant light intensity for several minutes; this eliminates the need for an automatic injection luminometer. The detection sensitivity of this method is of the order of 10 ^~ 20 moles of luciferase. This test is therefore approximately 100 times more sensitive than the CAT assay.

Experimental protocol:

The luciferase assay is carried out on a calibrated luminometer (model: BIOORBIT 1250). One hundred μl of reagent (Luciferase assay reagent- Promega) are vigorously mixed with the 20 μl of the sample to be assayed. The counting is carried out 10 seconds then 20 seconds after. The results are expressed in mVolts (De Wet et al., 1987).

6) Preparation of cell extracts

CMT cells are transfected according to the DEAE technique described by Ishikawa et al., 1992. Briefly, the cells are seeded at 2 10 "cells / dish 10 cm in diameter. Twenty-four hours later, the cells are washed twice. times with PBS, then 500 μl of trypsin are added The cells are incubated for 10 min at room temperature, then 4 ml of culture medium, containing 20 μg of the ER expression vector, 400 μg of DEAE dextran and 0.1 mM chloroquine is added The cells are incubated for 4 hours at 37 ° C followed by a 2 min shock with DMSO (dimethyl sulfoxide). One hour before harvesting the cells, estradiol (10 ^" ^ M) is added. The cells are washed twice with cold PBS, then harvested in the binding buffer (20 mM Tris-HCl (pH 7.5), DTT 2mM, 20% glycerol (v / v) and 0.4 M KC1. Total cell extracts are prepared by freezing the cells at -80 ° C. and then heating them on ice. This is followed by centrifugation at 10000 g for 15 min at 0 ° C. in a Sigma centrifuge The supernatant is stored at -80 ° C.

ER was also expressed in SF9 cells using the kit

"pBlueBac-His baculovirus expression System" purchased from Invitrog n.

The ER was prepared using the in vitro transcription and translation kit in a reticulocyte system (TNT -Promega). Briefly, 4 μg of ER expression vector is incubated with the TNT lysate, the T7 RNA polymerase, the mixture of amino acids (1 mM), and the RNAsin (40 IU). The reaction is incubated for two hours at 30 ° C.

7) Gel retardation experiments (EMSA):

The oligonucleotides are hybrid then radiolabeled using the Klenow fragment of DNA polymerase I. The tests are carried out as described by Cao et al., 1993. The binding reactions were carried out in binding buffer (20 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol (vol / vol), bovine albumin 3 μg / μl, 100 mM NaCl), 0.3 ng of radiolabelled DNA and lμg of dldC. In the case of baculovirus extracts enriched with ER, Nonidet P40 (Sigma) 0.1% is added to the reaction in order to minimize the binding of accessory proteins to the ER. Finally, the ER is added to the concentrations indicated in the legend figures. Thirty minutes after incubation at room temperature, the reaction mixture is deposited on an electrophoresis gel consisting of polyacrylamide 6% (acrylamide / bisacrylamide, 29: 1) and TBE 0.25 X, preheated (100 V / 12 cm , 30 min); migration continues for 90 min (200 V / 12 cm). The gel is then dried and then exposed with a film. The monoclonal antibody, H222, is incubated with the receptor during the binding reaction. The densitometric study is carried out using NIH Image software.

8) Methylation interference experiment:

The experiment is carried out as described by Truss et al. , 1991. Briefly, the oligonucleotides labeled with T4 polynucleotide kinase are treated with dimethyl sulfate (DMS). The methylated DNA is then added to 15 μl of ER purified recombinant (purchased from PanVera). The reaction mixture is then deposited on an electrophoresis gel composed of 6% acrylamide. After the self-radiography, the interesting bands are excised and then eluted overnight at 37 ° C. in 500 μl of buffer (Tris-EDTA, 1.5 M NaCl). After washing with phenol and chloroform and precipitation with ethanol, the DNA is cleaved with piperidine and then deposited on a denaturing gel of 10% polyacrylamide-8M urea. The gel is then dried and then autoradiographed.

9) Calculation of the cooperative relationship:

The equations described by Martinez et al (22) have been adapted for the sequence over ERE. The following model for the interaction of ER with the sequence over ERE is proposed:

(HA)

σm) complexes II and complexes IV are formed respectively by the dimer and the tetramer of ER linked to the sequence over ERE.

(ovERE) (ER2) (ovERE) (ER2) 2 (ovERE) (ER2) κdπ =

(HA) (IIB) (H) like the overe sequence is formed by two identical EREs:

(ΠA) = (ΠB) = I / 2 (Π)

(H) (ER2)

KdIV =

2 (IV)

Kdïï 4 (ovERE) (IV)

Cooperativity report = = -

KdIV ² (H) The radioactivity contained in complexes IV and II is determined by cutting the corresponding bands and counting them using a scintillation counter.

II) RESULTS

1) Linking the ER to the sequence over ERE

A new DNA sequence, called over ERE, consisting of two overlapping EREs, has been developed. This overlap was possible because the ERE consists of two half-sites separated by 3 bp. These three nucleotides have been transformed to create two identical overlapping EREs.

In order to determine whether this sequence binds the ER, gel retardation experiments were performed using the ERE and over ERE sequences. In these experiments, extracts of baculovirus infected cells enriched in ER and purified on a DEAE-dextran column were used. In the case of the ERE sequence, two bands are observed; these bands probably correspond to proteolysis states different from the dimeric form of the ER. In the case of the sequence over ERE, three bands are observed: bands 1 and 2 are identical to those observed for the ERE. However, a third majority band, migrating more slowly, is observed only for the sequence over ERE. This complex could contain additional ER molecules. The addition of the antibody H222, directed against the C terminal end of the ER, caused an additional delay of the 3 bands. These results therefore show that the sequence over ERE binds the ER. Competition with a 1000-fold excess of the GRE sequence did not alter the binding of the ER to the over ERE sequence. On the other hand, a 1000-fold excess of the non-radiolabelled ERE sequence abolished the formation of the complex over ERE-ER. These results show that the link between the ER and the over ERE sequence is specific. Similar results are obtained using purified ER extracts.

As the over ERE sequence is composed of two EREs, it is possible that the slowly migrating complex, observed in the case of over ERE, would be formed of two dimers of ER. In order to know if the four half-sites of the sequence over ERE are occupied by the ER, experiments of interference by methylation were carried out. The radiolabelled DNA is partially methylated following treatment with DMS; a connection between the DNA and the ER is then carried out. After retardation on gel, the slowly migrating complex is excised from the gel, then the DNA is cleaved by piperidine. Finally, the DNA resulting from digestion is deposited on a sequence gel. The G in position 2 of the sequence (AGGTC (A / G) or the G on the opposite strand of C in position 5 are important for the formation of the DNA-ER complex. Conversely, the G located outside of the ERE half-site are not essential for binding. The G at position 3 partially interfered. In conclusion, these results show that each of the 4 half-sites of the sequence over ERE binds a monomer of ER; this translates the binding from an ER tetramer to the over ERE sequence.

2) The ER binds cooperatively to the sequence over ERE

The cooperative binding of ER to the over ERE sequence has been demonstrated by means of binding experiments between the radiolabelled over ERE sequence and increasing amounts of ER. The rapidly migrating complex, corresponding to the ER dimer, appears from the low concentrations of ER added (complex II). As soon as the amount of ER becomes greater, the slowly migrating complex (IV complex) appears, then gradually becomes the majority complex. The amounts of complexes II and IV and of free DNA were determined by means of the radioactivity present in each band. The amount of DNA present in complex II increases first, but never becomes more than 20% of the total radioactivity of DNA. Conversely, complex IV, present at higher concentrations, quickly becomes the predominant form of radioactivity. Using these results and the equations presented in the materials and methods, the dissociation constants (Kd) of complexes II and IV, were determined. Kdll is approximately 15 times higher than KdIV. Thus, the second dimer binds with better affinity, if the neighboring sequence is occupied by the first dimer. These binding characteristics show that the ER binds cooperatively to the over ERE sequence.

In order to compare the apparent affinities of binding of ER to the ERE or over ERE sequences, experiments on gel retardation binding between the radiolabelled over ERE sequence and the ER were performed, when increasing amounts of ERE or over ERE cold are added. The addition of an excess of oligonucleotide over cold ERE prevented the formation of the complex with an efficiency 3 times greater than that of the oligonucleotide ERE. 3) Functionality of the sequence over ERE:

It was then examined whether the over ERE sequence is functional in the context of the ΔMTV promoter. This promoter is derived from the MMTV promoter but it is deleted from the -190 / -88 fragment sensitive to induction by glucocorticoids (Umesono et al., 1988). The CAT activity of cells transfected with this plasmid is not regulated by oestradiol.

The HepG2 cells were cotransfected with increasing concentrations of ER expression vector (0 ng to 100 ng), and either by the plasmid ΔMTV-ERE-CAT, or by the plasmid ΔMTV- (ERE) 2-CAT containing two ERE in tandem, or finally by the plasmid ΔMTV-over ERE-CAT. The hormone, 17-β Estradiol (10 ^" ^ M), is added to the culture medium 24 hours later. The transcriptional activity of the plasmid ΔMTV-ERE-CAT increases then reaches a plateau at 30 ng of ER transfected. in the case of the plasmid ΔMTV- (ERE) 2-CAT, the shape of the curve is similar to the first, however the transcriptional activity is twice greater than the plasmid ΔMTV-ERE- CAT. For the plasmid ΔMTV-over ERE-CAT , the transcriptional activity is 3 to 4 times higher than that obtained for the plasmid ΔMTV-ERE-CAT These results show that two EREs linked in tandem have an additive transcriptional activity, while two overlapping EREs have a synergistic transcriptional activity.

The possibility that the elements of the ΔMTV-CAT promoter contribute to the properties of over ERE has been tested. The ERE and over ERE sequences have been subcloned upstream of the different promoter, that of thymidine kinase (Tk). The transcriptional activity of the over ERE sequence is 4 to 5 times greater than that of the ERE sequence. Once again, these results show a functional synergy between two overlapping EREs, regardless of the context of the promoter. Similar results were obtained in MCF-7 cells.

As two overlapping EREs have synergistic transcriptional activity and two adjacent EREs have additive activity, a new sequence has been constructed, (over ERE) 2, formed of two adjacent over EREs, separated by 21 bp (center to center). Transient transfection experiments have shown that the transcriptional activity of the sequence (over ERE) 2 is similar to that of the sequence over ERE. These results show that an ER tetramer linked to the over ERE sequence is sufficient to induce transcription in an optimal manner, at least under these conditions. 4) Effect of xenoestrogens

The ER is activated by estrogens as well as by many natural or chemical compounds having an estrogenomimetic activity. These compounds are called xenoestrogens. The response of the promoters containing the ERE and over ERE sequences was compared with various classes of xenoestrogens.

Phenol red, a known colorant mimicking the effect of estrogens, was added, at different concentrations, to HepG2 cells transfected either by the ERE plasmid or by the over ERE plasmid. The transcriptional activity of the over ERE sequence is 4 to 5 times higher than that of the over ERE sequence, and this for the same concentrations of added phenol red. Consequently, using the ERE sequence, we can detect the effect of phenol red at a concentration of 10 ^~ 4 M, while the over ERE sequence allows us to detect the estrogenic activity of this compound at a lower concentration. (10 ^"6 M).

The estrogenic activity of a compound formed from two polychlorinated phenolic nuclei (Chlordane) was tested using the same test as that described for phenol red. Chlordane activated the transcription of the plasmid ERE-CAT at a concentration of 10 ^~ 5 M. On the other hand, one can detect the estrogenic activity of this molecule at a concentration of 10 ^" ^ M thanks to the plasmid over ERE-CAT In this case also the transcriptional activity of the sequence over ERE is very synergistic.

Therefore, synergistic activation of the transcription of the over ERE sequence is observed in the case of hormone hormone and of chemical compounds having an estrogenic activity.

5) Effect of antioestrogens:

The basal activity of the ΔMTV-over ERE-CAT plasmid is higher than that of the ΔMTV- (ERE) 2-CAT and ΔMTV-ERE-CAT plasmids. These differences in basal activities could be due either to the intrinsic properties of these promoters, or to an endogenous stimulation of the ER by compounds having an oestrogenomimetic activity. In order to test the second hypothesis, the HepG2 cells were transfected with one of these three plasmids mentioned above and then incubated either with oestradiol or with an ER antagonist ICI 168134 or with both. As expected, ICI 168134 strongly inhibited induction by esradiol, but this compound also inhibited the basal activity of these three constructions. These results show that the high basal activity of over ERE is due to endogenous stimulation of ER.

6) Areas A / B and E are responsible for the cooperative link of the ER to the sequence over ERE:

The cooperative binding of the ER to the over ERE sequence would be due either to an intrinsic property in the over ERE sequence, or to specific domains of the ER. In order to test the second hypothesis, truncated estradiol receptors were used. Cell extracts of CMT cells transfected either with one of the truncated ER expression vectors or with wild-type ER are incubated with the radiolabeled ERE or over ERE sequences. The migration of the complex containing the ERE dimer was determined using the ERE probe. Using the over ERE probe, wild ER (HEO) formed a tetrameric complex from the low ER concentrations added. This complex becomes predominant at higher ER concentrations. The cooperativity ratio (KdlI / KdIV) is close to 5, which indicates that the HEO binds cooperatively to the sequence over ERE. HE19, a truncated N-terminal receptor, is also capable of binding to the over ERE sequence in tetrameric form. Therefore the deletion of the A / B domain did not prevent the formation of the tetramer. However in this case, the cooperative relationship is close to 1, which suggests that this domain contributes in the cooperative link of the ER to the sequence over ERE. Several deletions of the C-terminal domain have been tested. HE13, a deleted F domain receptor has the same binding properties as HEO (KdII / KdIV = 4.9). Conversely, HE38, which is truncated from domain E, binds mainly in dimeric form. A weak band, observed only at high concentrations of added HE38, corresponds to the tetrameric form of the receptor. This was confirmed by the truncated HE15 receiver. When the C-terminal and N-terminal domains are deleted (HE70), no tetramer is observed, even if large quantities of HE70 are used, and when the majority of the probe is complexed. These results show that the C-terminal and N-terminal domains of ER are essential for the formation of the tetramer and for the cooperative binding of ER to the sequence over ERE.

The functionality of some of the deleted ER fragments has been tested. The HepG2 cells were transfected with either the Tk-ERE-CAT plasmid or the Tk-over ERE-CAT plasmid and increasing amounts of one of the deleted ER expression vectors. HEO and HE13 have the same induction profile, ie the transactivation performed by the over ERE sequence is approximately 5 times higher than that performed by the ERE sequence. In the case of HE19, the activation of the transcription carried out by the over ERE sequence is 2 to 3 times greater than that carried out by the ERE. So HE19 has lost the ability to perform synergistic transcriptional activation. HE 15 and HE21 have a very weak transcriptional activity and this for the two ERE or over ERE sequences. These results are in agreement with the gel retardation experiments, in which HEO and HE13 bind in a cooperative manner to the sequence over ERE. In conclusion, the cooperative binding of the estradiol receptor to the over ERE sequence in vitro correlates with the activation of synergistic transcription in vivo.

III) CONCLUSION

The ability of the estradiol receptor to bind to two overlapping estradiol response elements (over ERE) has been assessed. The binding and functional properties of the over ERE sequence have been studied. In addition to the dimeric complex, this sequence specifically binds a high molecular weight complex recognized by an antibody directed against ER. The structure of the over ERE sequence (4 half-sites) and the mobility of the complex show that it is a complex formed of a tetramer of ER linked to the over ERE sequence. This is reinforced by several arguments, (i) It is unlikely that the slowly migrating complex is formed by contamination of proteins which would delay the migration of the complex over ERE-ER, this because an identical migration profile is observed for different preparations of ER : ER prepared in cells infected with baculovirus, purified ER found on the market, ER prepared by an in vitro transcription / translation system, and finally cellular extracts of CMT cells transfected by ER. (ii) The methylation interference experiments have shown that the G residues located in the four ERE half-sites are totally or partially protected. However, the G residues located outside the ERE half-sites were not protected by the binding. As each ERE half site is occupied by an ER monomer, the over ERE sequence binds an ER tetramer.

The link of the ER to the sequence over ERE is cooperative. The dissociation constant (Kd) of the tetrameric complex (ER4-over ERE) is 5 to 15 times lower than that of the dimeric complex (ER2-over ERE), this depends on the extract used. These results show that the binding of the first dimer facilitates the binding of the second dimer, and this on either side of the DNA double helix.

Two possible mechanisms can lead to the cooperative nature of binding of the ER to the sequence over ERE: (i) Interactions between the receptors could stabilize the formation of the tetramer by increasing the affinity of the second dimer. (ii) The binding of the first dimer could modify the structure of DNA so as to increase the affinity of the second dimer. The use of receptor fragments made it possible to specify the mechanism involved. The deletion of the A / B domain did not prevent the formation of the ER-over ERE tetramer, but this altered the binding kinetics. Conversely, the deletion of domain E has given more striking results. In this case, the formation of the tetramer was severely impaired. When the two domains are deleted, the receptor therefore retains only the DNA binding domain, the binding of ER to sequences over ERE or ERE is identical, even at high ER concentrations. In contrast to the wild-type receptor, the binding of the binding domain to the DNA of the ER prevents the binding of a second dimer, the E domain is therefore critical. This domain would therefore help the DNA-receptor complex to get into a conformation which allows it to bind a second receptor dimer; this therefore constitutes an additional role for domain E.

The curvature of the DNA following interaction with the receptor could explain the observations mentioned above. Moreover, the binding of a dimer of the DNA binding domain of the ER is sufficient to induce a curvature of the DNA (Nardulli et al., 1993; Schwabe et al., 1993). This could prevent the binding of a DNA binding domain dimer at the overlapping site. The addition of the E domain makes it possible to overcome this inhibition, and the entire receptor binds cooperatively. As the E domain interacts with various proteins, the cooperative bond could be due to an interaction of the receptor-receptor type.

The ability to form a tetramer is not a unique property of ER. Another member of the nuclear receptor family, GR, is capable of forming a tetramer by interacting with overlapping GREs located in the promoter of the aspartate aminotransferase gene (Garlatti et al., 1994). The binding of the GR tetramer to overlapping response elements appears to be more cooperative than in the case of ER. This could be due to the fact that the GR does not curve the DNA following its binding (Luisi et al., 1991).

The two overlapping ERE sites in over ERE have synergistic transcriptional activity. The gel retardation binding experiments, as well as the transfection experiments carried out on wild ER and deleted, suggest that the cooperative link could explain the functional synergy observed ex vivo (Ptashne, 1986).

In conclusion, it is shown above that two elements of response to oestradiol can co-operatively bind a tetramer of ER, which leads to a strong hormonal induction. The formation of interaction between two receptor dimers during the binding on overlapping elements, reveals a good flexibility of the receptor molecule. It is possible that such interactions are brought into play between receptors binding to perfect half sites, or even to imperfect half sites, these interactions could stabilize this kind of DNA-ER complex. This would explain the effectiveness of these sequences in hormonal regulation.

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Claims

1. Use of nucleotide sequences comprising one or more response units to glucocorticoids, mineralocorticoids, androgens and progestins (also designated GRU), and/or one or more response units to estrogens (also designated ERU), said units being such that they understand :

- for the GRU unit: two nucleotide sequences, also called glucocorticoid response elements (or GRE elements), these two nucleotide sequences being derived by modification of one or more nucleotides of the GRE response element corresponding to the sequence d DNA of the following formula:

⁵ ' GGTACA XiX ₂ X ₃ TGTTCT ³ '

₃ _, _CCATGT _X ₄ X, X2, and X3, respectively,

- for the ERU unit: two nucleotide sequences, also called estrogen response elements (or ERE elements), these two nucleotide sequences being derived by modification of one or more nucleotides of the ERE response element corresponding to the sequence d DNA of the following formula:

⁵ ' AGGTCA X _! X ₂ X ₃ TGACCT ³ '

₃ _, _TCCACT _X ₄ one of these two elements is offset by a distance of 5 base pairs relative to the center of the other of these two elements, for the implementation of an in vitro detection or screening method of xenohormones, or for the preparation of drugs intended for the treatment of pathologies associated with the activity of glucocorticoid, mineralocorticoid, androgen and progestogen receptors, and/or estrogen receptors.

2. Use of nucleotide sequences according to claim 1, said nucleotide sequences being characterized in that they comprise as a GRU unit: - the following nucleotide sequence of formula (I):

_XalGT ACX _bl _GXciA _X _dl _GX _el _CCX _fl GTT _CX _gi ₃ _,

in which :

. the GRE response elements are indicated, on one of the DNA strands, in bold and italic characters,

. X _a to Xgi, independently of each other, represent any nucleotide or derivative, while X _a 2 to Xg2, represent a nucleotide or derivative capable of pairing with X _a ι to

- or a succession of at least two nucleotide sequences of formula (I) mentioned above, each of the nucleotide sequences of formula (I) being spaced by a distance of between lOn and l in base pairs, center to center, n being a integer greater than or equal to 2.

3. Use of nucleotide sequences according to claim 2, said nucleotide sequences being characterized in that:

- X _a ι, X51, Xf, and Xgi, independently of each other, represent any nucleotide _or derivative, while X _a 2 ^, X _a ι, X _D , Xf\, and Xgi, respectively,

- X _c ι represents A or T, while X _c 2 represents T or A respectively,

- Xdi represents A or C, while Xd2 represents T or G respectively,

- X _e represents A or T, while X _e 2 represents T or A respectively.

4. Use of nucleotide sequences according to one of claims 1 to 3, said nucleotide sequences being characterized in that they comprise as GRU unit:

- the following nucleotide sequence designated “GRE A”:

⁵ ' GGT ACA GAA AGA CCT GTT CT ³ ' 31 CCA TGT CTT TCT GGA CAA GA 51 in which the GRE response elements are indicated, on one of the DNA strands, in bold, italic, and underlined characters,

- or a succession of at least two GRE A nucleotide sequences, spaced at a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2, in particular: . the following sequence (GRE A)2:

'GGT ACA GAA AGA CCT GTT CTX _a GGT ACA GAA AGA CCT GTT CT ³ ', CCA TGT CTT TCT GGA CAA GAX _h CCA TGT CTT TCT GGA CAA GA ₅ ,

in which X _a represents any nucleotide or derivative, and X^ represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X^ represents G,

. the following sequence (GRE A)4:

' GGT ACA GAA AGA CCT GTT CTX _a GGT ACA GAA AGA CCT GTT CT ³ ' ι CCA TGT CTT TCT GGA CAA GAX _b CCA TGT CTT TCT GGA CAA GA ₅ . _' _{_} ^_ _{_} _{_} _{_}

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X y _, Xj, and _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

5. Use of nucleotide sequences according to one of claims 1 to 3, said nucleotide sequences being characterized in that they comprise as GRU unit:

- the following nucleotide sequence designated “GRE A up”:

⁵ ' GGT ACA GTA CGT CCT GTT CT ³ ' ₃ . CCA TGT CAT GCA GGA CAA GA 51

in which the GRE response elements are indicated, on one of the DNA strands, in bold, italic, and underlined characters, - or a succession of at least two GRE A up nucleotide sequences, spaced at a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2, in particular: . the following sequence (GRE A up)2:

' GGT ACA GTA CGT CCT GTT CTX _a GGT ACA GTA CGT CCT GTT CT ³ ' ι CCA TGT CAT GCA GGA CAA GAX _b CCA TGT CAT GCA GGA CAA GA ₅ .

in which X _a represents any nucleotide or derivative, and X represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X5 represents G,

. the following sequence (GRE A up)4:

' GGT ACA _GTA CGT CCT GTT CTX _a GGT ACA GTA CGT CCT GTT CT ³ ' 1 CCA TGT CAT GCA GGA CAA GAX _b CCA TGT CAT GCA GGA _CAA GA 5. ' CGT CCT GTT CT ³ ' 1 X _d CCA TGT CAT GCA GGA CAA GAX _f CCA TGT CAT GCA GGA CAA GA 51

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X _D _, Xj, and _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

6. Use of nucleotide sequences according to claim 1, said nucleotide sequences being characterized in that they comprise as an ERU unit:

- the following nucleotide sequence of formula (II):

X _al GG TCX _bl GGT CGA CCX _cl _GAC _CX _dx ₃ _,

in which :

. the ERE response elements are indicated, on one of the DNA strands, in bold and italic characters,

• al to ^dl _> independently of each other, represent any nucleotide or derivative, while X _a 2 to X<}2 _' represent a nucleotide or derivative capable of pairing with X _a ι to X^i respectively, - or a succession of at least two nucleotide sequences of formula (II) above, spaced at a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2.

7. Use of nucleotide sequences according to claim 6, said nucleotide sequences being characterized in that they comprise as an ERU unit:

- the following nucleotide sequence designated “over ERE”:

⁵ ' AGG TCA GGT CGA CCT GAC CT ³ ' ₃ . TCC AGT CCA GCT GGA CTG GA ₅ .

in which the ERE response elements are indicated, on one of the DNA strands, in bold, italic and underlined characters,

- or a succession of at least two nucleotide sequences over ERE, spaced at a distance between lOn and l in base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (over ERE)2:

⁵ ' AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ ' ₃ . TCC AGT CCA GCT GGA CTG GAX _b TCC AGT CCA GCT GGA CTG GA ₅ .

in which X _a represents any nucleotide or derivative, and X _D represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X represents G,

. the following sequence (over ERE)4:

⁵ ' AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ '

31 TCC AGT CCA GCT GGA CTG GAX TCC AGT CCA GCT GGA CTG GA 51

⁵ ' X _C AGG_TÇA GGT CGA CCT GAC CTX _e AGG TCA GGT CGA CCT GAC CT ³

3. X _d TCC AGT CCA GCT GGA CTG GAX _f TCC AGT CCA GCT GGA CTG GA 5

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X, X _^ , and _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

8. Nucleotide sequence characterized in that it comprises a succession of at least two GRE A nucleotide sequences as defined in claim 4, and/or a succession of at least two GRE A up nucleotide sequences as defined in claim 4. claim 5.

9. Nucleotide sequence characterized in that it comprises as an ERU unit:

- the following nucleotide sequence of formula (II):

X _al _GG TCX _bl _GGT CGA CCX _cl _GAC _CX _di

in which :

. X _a to Xdl, independently of each other, represent any nucleotide or derivative, while X _a 2 to Xj2 _> represent a nucleotide or derivative capable of pairing with X _a to

- or a succession of at least two nucleotide sequences of formula (II) above, spaced at a distance between lOn and One base pairs, center to center, n being an integer greater than or equal to 2.

10. Nucleotide sequence according to claim 9, characterized in that it comprises as an ERU unit:

- the following nucleotide sequence over ERE:

⁵ ' AGG TCA GGT CGA CCT GAC CT ³ ' 3. TCC AGT CCA GCT GGA CTG GA 51

- or a succession of at least two nucleotide sequences over ERE, spaced at a distance between lOn and One base pairs, center to center, n being an integer greater than or equal to 2, in particular:

. the following sequence (over ERE)2:

' AGG TCA GGT CGA CCT GAC CTX _Λ AGG TCA GGT CGA CCT GAC CT ³ ' 1 TCC AGT CCA GCT GGA CTG GAX _b TCC AGT CCA GCT GGA CTG GA 5. in which X _a represents any nucleotide or derivative, and X represents a nucleotide or derivative capable of pairing with X _a , in particular X _a represents C and X represents G,

. the following sequence (over ERE)4:

⁵ ' AGG TCA GGT CGA CCT GAC CTX _a AGG TCA GGT CGA CCT GAC CT ³ '

3. TCC AGT CCA GCT GGA CTG GAX _b TCC AGT CCA GCT GGA CTG GA 51

⁵ ' X _ç AGG TCA GGT CGA CCT GAC CTX _e AGG TCA GGT CGA CCT GAC CT ³ '

31 X _d TCC AGT CCA GCT GGA CTG GAX _f TCC AGT CCA GCT GGA CTG GA 51

in which X _a , X _c , and X _e , independently of each other, represent any nucleotide or derivative, while X^ _, Xςj, and _c , and X _e , respectively, in particular X _a , X _c , and X _e represent C and X _a , X _c , and X _e represent G.

11. Nucleotide sequence according to claim 9 or 10, characterized in that it comprises one or more GRU units defined in one of claims 1 to 5.

12. Nucleotide sequence according to one of claims 9 to 11, characterized in that the ERU unit(s), and, where appropriate the GRU unit(s), are located upstream of a promoter controlling the transcription of a gene rapporteur.

13. Vector, in particular plasmid, characterized in that it comprises a nucleotide sequence according to one of claims 9 to 12.

14. Human or animal cell transformed by a vector according to claim 13.

15. Method for in vitro detection or screening of xenohormones capable of interacting with glucocorticoid receptors, mineralocorticoids, androgens and progesterone, and/or estrogen receptors in the human body, characterized in that it comprises the culturing respectively of human or animal cells transformed by a vector comprising at least one GRU unit defined in one of the claims 1 to 5, and/or human or animal cells according to claim 14, in a culture medium in the presence of a biological sample capable of containing said xenohormones,

- detecting the presence of the polypeptide encoded by said reporter gene in said cells or the culture medium thereof, in particular by colorimetry in the case where the polypeptide encoded by the reporter gene reacts with its substrate as part of a reaction colored, or by fluorimetry in the case where the polypeptide encoded by the reporter gene is fluorescent, or capable of being recognized by a fluorescent molecule, or by a radioactive method.

16. Vector for gene therapy characterized in that it comprises at least one GRU unit defined in one of claims 1 to 5, and/or at least one ERU unit defined in one of claims 1, 6 and 7 .

17. Pharmaceutical composition characterized in that it comprises, in association with a physiologically acceptable vehicle, at least one GRU unit defined in one of claims 1 to 5, and/or at least one ERU unit defined in one of claims 1, 6 and 7, said units being where appropriate preceded and followed by a succession of at least approximately 5 nucleotides or derivatives, or a vector according to claim 16.